Nucleic acid analysis using sequence tokens

ABSTRACT

The present invention provides methods and compositions for tagging nucleic acid sequence fragments, e.g., a set of nucleic acid sequence fragments from a single genome, with one or more unique members of a collection of oligonucleotide tags, or sequence tokens, which, in turn, can be identified using a variety of readout platforms. As a general rule, a given sequence token is used once and only once in any tag sequence. In addition, the present invention also provides methods for using the sequence tokens to efficiently determine variations in nucleotide sequences in the associated nucleic acid sequence fragments.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.60/761,577 filed Jan. 23, 2006, which application is incorporated hereinby reference.

BACKGROUND

There is great interest in determining nucleic acid sequences andsequence differences rapidly and efficiently for addressing a host ofimportant problems in the biomedical sciences, e.g. Collins et al,Nature, 422: 835-847 (2003); National Cancer Institute, Report ofWorking Group on Biomedical Technology, “Recommendation for a HumanCancer Genome Project,” (February, 2005). Not only are such measurementscrucial for understanding the genetic basis of inherited traits, such asdisease susceptibilities, but they are also crucial for understandingthe role of somatic mutations in cancer. Many techniques have beendeveloped and successfully applied to problems in these areas, e.g.Stephens et al, Nature Genetics, 37: 590-592 (2005); Syvanen, NatureReviews Genetics, 2: 930-942 (2002); Kennedy et al, NatureBiotechnology, 21: 1233-1237 (2003); Hardenbol et al, Genome Research,15: 269-275 (2005); Gunderson et al, Nature Genetics, 37: 549-554(2005); Margulies et al, Nature, 437: 376-380 (2005); and the like.However, there are still many problems, such as the rapid and efficientdiscovery of genetic or epigenetic variation, that are not adequatelyaddressed by current techniques.

Among current techniques, several have employed oligonucleotide tags, orbarcodes, to represent and/or convey target sequence information fromknown sequences, e.g. Hardenbol et al (cited above); Shoemaker et al,Nature Genetics, 14: 450-456 (1996); Gerry et al, J. Mol. Biol., 292:251-262 (1999). The use of such reagents is advantageous because theycan be selected to maximize convenience and efficiency ofidentification, such as by selective and specific hybridization tocomplementary sequences on a microarray for a parallel readout ofsequence information. However, such techniques presently measure onlyknown mutations or polymorphisms and require large-scale synthesis ofprobes and tags prior to application.

The availability of a convenient and efficient molecular tagging methodthat could be used for discovery of genetic variation and/orhigh-throughput DNA sequencing would extend the use of these usefulreagents and lead to improvements in analytical assays in many fields,including scientific and biomedical research, medicine, and otherindustrial areas where genetic measurements are important.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for taggingnucleic acid sequence fragments, e.g., a set of nucleic acid sequencefragments from a single genome, with one or more unique members of acollection of oligonucleotide tags, or sequence tokens, which, in turn,can be identified using a variety of readout platforms. As a generalrule, a given sequence token is used once and only once in any tagsequence. In addition, the present invention also provides methods forusing the sequence tokens to efficiently determine variations innucleotide sequences in the associated nucleic acid sequence fragments.

The present invention provides a method of tagging a plurality ofpopulations of polynucleotides each with a unique sequence token bygenerating a plurality of unique sequence token tags; dividingpolynucleotides of each population into at least two non-overlappingnucleic acid segments; and ligating a unique sequence token tag to eachnon-overlapping nucleic acid segment of each population ofpolynucleotides to provide a plurality of populations of polynucleotideseach tagged with a unique sequence token tag, wherein each uniquesequence token tag is used to tag only one population ofpolynucleotides.

In some embodiments, the predetermined number of the non-overlappingsegments is all non-overlapping segments of the polynucleotide. In someembodiments, each of the non-overlapping segments has a length that isthe same for all such segments. In some embodiments, each population ofpolynucleotides includes genomic DNA from a single subject, such as ahuman. In some embodiments, the dividing is by restriction enzymedigestion of the polynucleotides.

The present invention also provides a method of tagging a plurality ofgenomic DNA samples from a plurality of subjects each with a uniquesequence token by generating a plurality of unique sequence token tags;dividing each genomic DNA sample from each subjects into at least twonon-overlapping nucleic acid segments; and ligating a unique sequencetoken tag to each non-overlapping nucleic acid segment of each genomicDNA sample to provide a plurality of genomic DNA samples from aplurality of subjects each tagged with a unique sequence token tag,wherein each unique sequence token tag is used to tag only the genomicDNA from one subject.

In some embodiments, the predetermined number of the non-overlappingsegments is all non-overlapping segments of the polynucleotide. In someembodiments, each of the non-overlapping segments has a length that isthe same for all such segments. In some embodiments, the subject is ahuman. In some embodiments, the dividing is by restriction enzymedigestion of the polynucleotides.

The present invention also provides a method of screening for thepresence or absence of a rare nucleotide allele of a polymorphism in apopulation of enriched genomic DNA segments from a population of genomicDNA samples, by incubating a reaction mixture under polymerizationconditions, including: an enriched population of non-overlapping genomicDNA segments, wherein each genomic DNA segment comprises a uniquesequence token, an oligonucleotide probe complementary to a region ofthe genomic DNA segment upstream of a polymorphism, wherein thepolymorphism comprises a rare nucleotide allele and a frequentnucleotide allele, and dideoxy nucleotide triphosphate corresponding tothe rare nucleotide allele and the frequent nucleotide allele, whereinthe nucleotide triphosphate corresponding to the rare nucleotide alleleis conjugated to a first member of a binding pair; dividing the reactionmixture into at least two groups by exposing the reaction mixture to thesecond member of the binding pair to provide a first group comprisingoligonucleotide probes having the nucleotide triphosphate correspondingto the rare nucleotide hybridized to the genomic DNA segments to providea group of bound genomic DNA segments; and determining the uniquesequence tokens of the bound genomic DNA segments to identify genomicDNA samples having the rare nucleotide allele of the polymorphism.

In some embodiments, the predetermined number of the non-overlappingsegments is all non-overlapping segments of the polynucleotide. In someembodiments, the first binding member is biotin, avidin, strepavidin, ora magnetic bead. In some embodiments, the second binding member isbiotin, avidin, strepavidin, or a magnetic bead. In some embodiments,the step of determining includes specifically hybridizing said generatedsequence tokens with complements thereof attached to one or more solidphase supports. In some embodiments, the step of determining includessequencing the sequence token or a portion thereof.

The present invention also provides a sieving device, including: atleast one substrate support having an bottom surface and a top surface,wherein the bottom surface comprises a plurality linear elements andwherein each linear element comprises a unique oligonucleotideimmobilized thereon; and a receiving unit having top surface comprisinga microfluidic channel, wherein the bottom surface of the substrate ispositioned on the top surface of the receiving unit and the linearelements of the substrate extend into the microfluidic channel. In someembodiments, the device further includes a plurality of substratesupports.

The present invention also includes a kit including: a sieving devicecomprising at least one substrate having a top surface and a bottomsurface, wherein the bottom surface comprises a plurality linearelements and wherein each linear element comprises a uniqueoligonucleotide immobilized thereon, and a receiving unit having topsurface comprising a microfluidic channel, wherein the bottom surface ofthe substrate is positioned on the top surface of the receiving unit andthe linear elements of the substrate extend into the microfluidicchannel; and instructions for using the sieving device to identifysequence tokens in a population. In some embodiments, the kit furtherincludes a plurality of substrate supports.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the invention as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIGS. 1A-1B illustrate a general procedure for attaching anoligonucleotide tag to one end of a polynucleotide.

FIG. 2 shows a schematic of an exemplary tagged genomic DNA fragmentpanel A and an exemplary concatenation process of sequence unique tokensin panel B.

FIG. 3 provides a schematic diagram of a method for identifyingindividuals or samples carrying a rare allele by using the sequencetoken tagging system.

FIG. 4 provides a schematic depiction of how the tagged nucleic acidpopulation can first be separated into frequent allele groups prior toanalysis of the presence or absence of a rare allele.

FIG. 5 provides a schematic diagram of a method for identifyingindividuals or samples carrying a rare allele by using the sequencetoken tagging system and wild type RNA probes.

FIG. 6 shows schematic diagrams of an exemplary sieving device. Panel Ashows arrangement of the linear elements, or pins, each having ananti-sequence token immobilized thereon and arranged in a comb-likemanner. Panel B shows the bottom view a series of the combs arranges ina block. Panes C and D show cross-sections of a comb placed in a channelwhere the immobilized anti-sequence token can come into contact with thepopulation of sequence tokens.

DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Still, certain elements aredefined for the sake of clarity and ease of reference.

Terms and symbols of nucleic acid chemistry, biochemistry, genetics, andmolecular biology used herein follow those of standard treatises andtexts in the field, e.g. Kornberg and Baker, DNA Replication, SecondEdition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, SecondEdition (Worth Publishers, New York, 1975); Strachan and Read, HumanMolecular Genetics, Second Edition (Wiley-Liss, New York, 1999);Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach(Oxford University Press, New York, 1991); Gait, editor, OligonucleotideSynthesis: A Practical Approach (IRL Press, Oxford, 1984); and the like.

“Addressable” in reference to tag complements means that the nucleotidesequence, or perhaps other physical or chemical characteristics, of anend-attached probe, such as a tag complement, can be determined from itsaddress, i.e. a one-to-one correspondence between the sequence or otherproperty of the end-attached probe and a spatial location on, orcharacteristic of, the solid phase support to which it is attached.Preferably, an address of a tag complement is a spatial location, e.g.the planar coordinates of a particular region containing copies of theend-attached probe. However, end-attached probes may be addressed inother ways too, e.g. by microparticle size, shape, color, frequency ofmicro-transponder, or the like, e.g. Chandler et al, PCT publication WO97/14028.

“Amplicon” means the product of a polynucleotide amplification reaction.That is, it is a population of polynucleotides, usually double stranded,that are replicated from one or more starting sequences. The one or morestarting sequences may be one or more copies of the same sequence, or itmay be a mixture of different sequences. Amplicons may be produced by avariety of amplification reactions whose products are multiplereplicates of one or more target nucleic acids. Generally, amplificationreactions producing amplicons are “template-driven” in that base pairingof reactants, either nucleotides or oligonucleotides, have complementsin a template polynucleotide that are required for the creation ofreaction products. In one aspect, template-driven reactions are primerextensions with a nucleic acid polymerase or oligonucleotide ligationswith a nucleic acid ligase. Such reactions include, but are not limitedto, polymerase chain reactions (PCRs), linear polymerase reactions,nucleic acid sequence-based amplification (NASBAs), rolling circleamplifications, and the like, disclosed in the following references thatare incorporated herein by reference: Mullis et al, U.S. Pat. Nos.4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al, U.S.Pat. No. 5,210,015 (real-time PCR with “TAQMAN™” probes); Wittwer et al,U.S. Pat. No. 6,174,670; Kacian et al, U.S. Pat. No. 5,399,491(“NASBA”); Lizardi, U.S. Pat. No. 5,854,033; Aono et al, Japanese patentpubl. JP 4-262799 (rolling circle amplification); and the like. In oneaspect, amplicons of the invention are produced by PCRs. Anamplification reaction may be a “real-time” amplification if a detectionchemistry is available that permits a reaction product to be measured asthe amplification reaction progresses, e.g. “real-time PCR” describedbelow, or “real-time NASBA” as described in Leone et al, Nucleic AcidsResearch, 26: 2150-2155 (1998), and like references. As used herein, theterm “amplifying” means performing an amplification reaction. A“reaction mixture” means a solution containing all the necessaryreactants for performing a reaction, which may include, but not belimited to, buffering agents to maintain pH at a selected level during areaction, salts, co-factors, scavengers, and the like.

The term “assessing” includes any form of measurement, and includesdetermining if an element is present or not. The terms “determining”,“measuring”, “evaluating”, “assessing” and “assaying” are usedinterchangeably and includes quantitative and qualitativedeterminations. Assessing may be relative or absolute. “Assessing thepresence of” includes determining the amount of something present,and/or determining whether it is present or absent. As used herein, theterms “determining,” “measuring,” and “assessing,” and “assaying” areused interchangeably and include both quantitative and qualitativedeterminations.

“Complementary or substantially complementary” refers to thehybridization or base pairing or the formation of a duplex betweennucleotides or nucleic acids, such as, for instance, between the twostrands of a double stranded DNA molecule or between an oligonucleotideprimer and a primer binding site on a single stranded nucleic acid.Complementary nucleotides are, generally, A and T (or A and U), or C andG. Two single stranded RNA or DNA molecules are said to be substantiallycomplementary when the nucleotides of one strand, optimally aligned andcompared and with appropriate nucleotide insertions or deletions, pairwith at least about 80% of the nucleotides of the other strand, usuallyat least about 90% to 95%, and more preferably from about 98 to 100%.Alternatively, substantial complementarity exists when an RNA or DNAstrand will hybridize under selective hybridization conditions to itscomplement. Typically, selective hybridization will occur when there isat least about 65% complementary over a stretch of at least 14 to 25nucleotides, preferably at least about 75%, more preferably at leastabout 90% complementary. See, M. Kanehisa Nucleic Acids Res. 12:203(1984), incorporated herein by reference.

“Duplex” means at least two oligonucleotides and/or polynucleotides thatare fully or partially complementary undergo Watson-Crick type basepairing among all or most of their nucleotides so that a stable complexis formed. The terms “annealing” and “hybridization” are usedinterchangeably to mean the formation of a stable duplex. “Perfectlymatched” in reference to a duplex means that the poly- oroligonucleotide strands making up the duplex form a double strandedstructure with one another such that every nucleotide in each strandundergoes Watson-Crick basepairing with a nucleotide in the otherstrand. The term “duplex” comprehends the pairing of nucleoside analogs,such as deoxyinosine, nucleosides with 2-aminopurine bases, PNAs, andthe like, that may be employed. A “mismatch” in a duplex between twooligonucleotides or polynucleotides means that a pair of nucleotides inthe duplex fails to undergo Watson-Crick bonding.

“Genetic locus,” or “locus” in reference to a genome or targetpolynucleotide, means a contiguous subregion or segment of the genome ortarget polynucleotide. As used herein, genetic locus, or locus, mayrefer to the position of a nucleotide, a gene, or a portion of a gene ina genome, including mitochondrial DNA, or it may refer to any contiguousportion of genomic sequence whether or not it is within, or associatedwith, a gene. In one aspect, a genetic locus refers to any portion ofgenomic sequence, including mitochondrial DNA, from a single nucleotideto a segment of few hundred nucleotides, e.g. 100-300, in length.

“Genetic variant” means a substitution, inversion, insertion, ordeletion of one or more nucleotides at genetic locus, or a translocationof DNA from one genetic locus to another genetic locus. In one aspect,genetic variant means an alternative nucleotide sequence at a geneticlocus that may be present in a population of individuals and thatincludes nucleotide substitutions, insertions, and deletions withrespect to other members of the population. In another aspect,insertions or deletions at a genetic locus comprises the addition or theabsence of from 1 to 10 nucleotides at such locus, in comparison withthe same locus in another individual of a population.

“Kit” refers to any delivery system for delivering materials or reagentsfor carrying out a method of the invention. In the context of reactionassays, such delivery systems include systems that allow for thestorage, transport, or delivery of reaction reagents (e.g., probes,enzymes, etc. in the appropriate containers) and/or supporting materials(e.g., buffers, written instructions for performing the assay etc.) fromone location to another. For example, kits include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. Such contents may be delivered to theintended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains probes.

“Ligation” means to form a covalent bond or linkage between the terminiof two or more nucleic acids, e.g. oligonucleotides and/orpolynucleotides, in a template-driven reaction. The nature of the bondor linkage may vary widely and the ligation may be carried outenzymatically or chemically. As used herein, ligations are usuallycarried out enzymatically to form a phosphodiester linkage between a 5′carbon of a terminal nucleotide of one oligonucleotide with 3′ carbon ofanother oligonucleotide. A variety of template-driven ligation reactionsare described in the following references, which are incorporated byreference: Whitely et al, U.S. Pat. No. 4,883,750; Letsinger et al, U.S.Pat. No. 5,476,930; Fung et al, U.S. Pat. No. 5,593,826; Kool, U.S. Pat.No. 5,426,180; Landegren et al, U.S. Pat. No. 5,871,921; Xu and Kool,Nucleic Acids Research, 27: 875-881 (1999); Higgins et al, Methods inEnzymology, 68: 50-71 (1979); Engler et al, The Enzymes, 15: 3-29(1982); and Namsaraev, U.S. patent publication 2004/0110213.

“Microarray” refers to a solid phase support having a planar surface,which carries an array of nucleic acids, each member of the arraycomprising identical copies of an oligonucleotide or polynucleotideimmobilized to a spatially defined region or site, which does notoverlap with those of other members of the array; that is, the regionsor sites are spatially discrete. Spatially defined hybridization sitesmay additionally be “addressable” in that its location and the identityof its immobilized oligonucleotide are known or predetermined, forexample, prior to its use. Typically, the oligonucleotides orpolynucleotides are single stranded and are covalently attached to thesolid phase support, usually by a 5′-end or a 3′-end. The density ofnon-overlapping regions containing nucleic acids in a microarray istypically greater than 100 per cm², and more preferably, greater than1000 per cm². Microarray technology is reviewed in the followingreferences: Schena, Editor, Microarrays: A Practical Approach (IRLPress, Oxford, 2000); Southern, Current Opin. Chem. Biol., 2: 404-410(1998); Nature Genetics Supplement, 21: 1-60 (1999). As used herein,“random microarray” refers to a microarray whose spatially discreteregions of oligonucleotides or polynucleotides are not spatiallyaddressed. That is, the identity of the attached oligonucleoties orpolynucleotides is not discernable, at least initially, from itslocation. In one aspect, random microarrays are planar arrays ofmicrobeads wherein each microbead has attached a single kind ofhybridization tag complement, such as from a minimally cross-hybridizingset of oligonucleotides. Arrays of microbeads may be formed in a varietyof ways, e.g. Brenner et al, Nature Biotechnology, 18: 630-634 (2000);Tulley et al, U.S. Pat. No. 6,133,043; Stuelpnagel et al, U.S. Pat. No.6,396,995; Chee et al, U.S. Pat. No. 6,544,732; and the like. Likewise,after formation, microbeads, or oligonucleotides thereof, in a randomarray may be identified in a variety of ways, including by opticallabels, e.g. fluorescent dye ratios or quantum dots, shape, sequenceanalysis, or the like.

“Nucleoside” as used herein includes the natural nucleosides, including2′-deoxy and 2′-hydroxyl forms, e.g. as described in Kornberg and Baker,DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). “Analogs” inreference to nucleosides includes synthetic nucleosides having modifiedbase moieties and/or modified sugar moieties, e.g. described by Scheit,Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman,Chemical Reviews, 90: 543-584 (1990), or the like, with the proviso thatthey are capable of specific hybridization. Such analogs includesynthetic nucleosides designed to enhance binding properties, reducecomplexity, increase specificity, and the like. Polynucleotidescomprising analogs with enhanced hybridization or nuclease resistanceproperties are described in Uhlman and Peyman (cited above); Crooke etal, Exp. Opin. Ther. Patents, 6: 855-870 (1996); Mesmaeker et al,Current Opinion in Structual Biology, 5: 343-355 (1995); and the like.Exemplary types of polynucleotides that are capable of enhancing duplexstability include oligonucleotide N3′→P5′ phosphoramidates (referred toherein as “amidates”), peptide nucleic acids (referred to herein as“PNAs”), oligo-2′-O-alkylribonucleotides, polynucleotides containing C-5propynylpyrimidines, locked nucleic acids (LNAs), and like compounds.Such oligonucleotides are either available commercially or may besynthesized using methods described in the literature.

“Polymerase chain reaction,” or “PCR,” means a reaction for the in vitroamplification of specific DNA sequences by the simultaneous primerextension of complementary strands of DNA. In other words, PCR is areaction for making multiple copies or replicates of a target nucleicacid flanked by primer binding sites, such reaction comprising one ormore repetitions of the following steps: (i) denaturing the targetnucleic acid, (ii) annealing primers to the primer binding sites, and(iii) extending the primers by a nucleic acid polymerase in the presenceof nucleoside triphosphates. Usually, the reaction is cycled throughdifferent temperatures optimized for each step in a thermal cyclerinstrument. Particular temperatures, durations at each step, and ratesof change between steps depend on many factors well-known to those ofordinary skill in the art, e.g. exemplified by the references: McPhersonet al, editors, PCR: A Practical Approach and PCR2: A Practical Approach(IRL Press, Oxford, 1991 and 1995, respectively). For example, in aconventional PCR using Taq DNA polymerase, a double stranded targetnucleic acid may be denatured at a temperature >90° C., primers annealedat a temperature in the range 50-75° C., and primers extended at atemperature in the range 72-78° C. The term “PCR” encompasses derivativeforms of the reaction, including but not limited to, RT-PCR, real-timePCR, nested PCR, quantitative PCR, multiplexed PCR, and the like.Reaction volumes range from a few hundred nanoliters, e.g. 200 nL, to afew hundred μL, e.g. 200 μL. “Reverse transcription PCR,” or “RT-PCR,”means a PCR that is preceded by a reverse transcription reaction thatconverts a target RNA to a complementary single stranded DNA, which isthen amplified, e.g. Tecott et al, U.S. Pat. No. 5,168,038, which patentis incorporated herein by reference. “Real-time PCR” means a PCR forwhich the amount of reaction product, i.e. amplicon, is monitored as thereaction proceeds. There are many forms of real-time PCR that differmainly in the detection chemistries used for monitoring the reactionproduct, e.g. Gelfand et al, U.S. Pat. No. 5,210,015 (“taqman”); Wittweret al, U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes);Tyagi et al, U.S. Pat. No. 5,925,517 (molecular beacons); which patentsare incorporated herein by reference. Detection chemistries forreal-time PCR are reviewed in Mackay et al, Nucleic Acids Research, 30:1292-1305 (2002), which is also incorporated herein by reference.“Nested PCR” means a two-stage PCR wherein the amplicon of a first PCRbecomes the sample for a second PCR using a new set of primers, at leastone of which binds to an interior location of the first amplicon. Asused herein, “initial primers” in reference to a nested amplificationreaction mean the primers used to generate a first amplicon, and“secondary primers” mean the one or more primers used to generate asecond, or nested, amplicon. “Multiplexed PCR” means a PCR whereinmultiple target sequences (or a single target sequence and one or morereference sequences) are simultaneously carried out in the same reactionmixture, e.g. Bernard et al, Anal. Biochem., 273: 221-228(1999)(two-color real-time PCR). Usually, distinct sets of primers areemployed for each sequence being amplified.

“Quantitative PCR” means a PCR designed to measure the abundance of oneor more specific target sequences in a sample or specimen. QuantitativePCR includes both absolute quantitation and relative quantitation ofsuch target sequences. Quantitative measurements are made using one ormore reference sequences that may be assayed separately or together witha target sequence. The reference sequence may be endogenous or exogenousto a sample or specimen, and in the latter case, may comprise one ormore competitor templates. Typical endogenous reference sequencesinclude segments of transcripts of the following genes: β-actin, GAPDH,β₂-microglobulin, ribosomal RNA, and the like. Techniques forquantitative PCR are well-known to those of ordinary skill in the art,as exemplified in the following references that are incorporated byreference: Freeman et al, Biotechniques, 26: 112-126 (1999);Becker-Andre et al, Nucleic Acids Research, 17: 9437-9447 (1989);Zimmerman et al, Biotechniques, 21: 268-279 (1996); Diviacco et al,Gene, 122: 3013-3020 (1992); Becker-Andre et al, Nucleic Acids Research,17: 9437-9446 (1989); and the like.

“Polynucleotide” or “oligonucleotide” are used interchangeably and eachmean a linear polymer of nucleotide monomers. Monomers making uppolynucleotides and oligonucleotides are capable of specifically bindingto a natural polynucleotide by way of a regular pattern ofmonomer-to-monomer interactions, such as Watson-Crick type of basepairing, base stacking, Hoogsteen or reverse Hoogsteen types of basepairing, or the like. Such monomers and their internucleosidic linkagesmay be naturally occurring or may be analogs thereof, e.g. naturallyoccurring or non-naturally occurring analogs. Non-naturally occurringanalogs may include PNAs, phosphorothioate internucleosidic linkages,bases containing linking groups permitting the attachment of labels,such as fluorophores, or haptens, and the like. Whenever the use of anoligonucleotide or polynucleotide requires enzymatic processing, such asextension by a polymerase, ligation by a ligase, or the like, one ofordinary skill would understand that oligonucleotides or polynucleotidesin those instances would not contain certain analogs of internucleosidiclinkages, sugar moities, or bases at any or some positions.Polynucleotides typically range in size from a few monomeric units, e.g.5-40, when they are usually referred to as “oligonucleotides,” toseveral thousand monomeric units. Whenever a polynucleotide oroligonucleotide is represented by a sequence of letters (upper or lowercase), such as “ATGCCTG,” it will be understood that the nucleotides arein 5′→3′ order from left to right and that “A” denotes deoxyadenosine,“C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotesthymidine, “I” denotes deoxyinosine, “U” denotes uridine, unlessotherwise indicated or obvious from context. Unless otherwise noted theterminology and atom numbering conventions will follow those disclosedin Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York,1999). Usually polynucleotides comprise the four natural nucleosides(e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine forDNA or their ribose counterparts for RNA) linked by phosphodiesterlinkages; however, they may also comprise non-natural nucleotideanalogs, e.g. including modified bases, sugars, or internucleosidiclinkages. It is clear to those skilled in the art that where an enzymehas specific oligonucleotide or polynucleotide substrate requirementsfor activity, e.g. single stranded DNA, RNA/DNA duplex, or the like,then selection of appropriate composition for the oligonucleotide orpolynucleotide substrates is well within the knowledge of one ofordinary skill, especially with guidance from treatises, such asSambrook et al, Molecular Cloning, Second Edition (Cold Spring HarborLaboratory, New York, 1989), and like references.

“Primer” means an oligonucleotide, either natural or synthetic, that iscapable, upon forming a duplex with a polynucleotide template, of actingas a point of initiation of nucleic acid synthesis and being extendedfrom its 3′ end along the template so that an extended duplex is formed.The sequence of nucleotides added during the extension process aredetermined by the sequence of the template polynucleotide. Usuallyprimers are extended by a DNA polymerase. Primers are generally of alength compatible with its use in synthesis of primer extensionproducts, and are usually are in the range of between 8 to 100nucleotides in length, such as 10 to 75, 15 to 60, 15 to 40, 18 to 30,20 to 40, 21 to 50, 22 to 45, 25 to 40, and so on, more typically in therange of between 18-40, 20-35, 21-30 nucleotides long, and any lengthbetween the stated ranges. Typical primers can be in the range ofbetween 10-50 nucleotides long, such as 15-45, 18-40, 20-30, 21-25 andso on, and any length between the stated ranges. In some embodiments,the primers are usually not more than about 10, 12, 15, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70nucleotides in length.

Primers are usually single-stranded for maximum efficiency inamplification, but may alternatively be double-stranded. Ifdouble-stranded, the primer is usually first treated to separate itsstrands before being used to prepare extension products. Thisdenaturation step is typically effected by heat, but may alternativelybe carried out using alkali, followed by neutralization. Thus, a“primer” is complementary to a template, and complexes by hydrogenbonding or hybridization with the template to give a primer/templatecomplex for initiation of synthesis by a polymerase, which is extendedby the addition of covalently bonded bases linked at its 3′ endcomplementary to the template in the process of DNA synthesis.

A “primer pair” as used herein refers to first and second primers havingnucleic acid sequence suitable for nucleic acid-based amplification of atarget nucleic acid. Such primer pairs generally include a first primerhaving a sequence that is the same or similar to that of a first portionof a target nucleic acid, and a second primer having a sequence that iscomplementary to a second portion of a target nucleic acid to providefor amplification of the target nucleic acid or a fragment thereof.Reference to “first” and “second” primers herein is arbitrary, unlessspecifically indicated otherwise. For example, the first primer can bedesigned as a “forward primer” (which initiates nucleic acid synthesisfrom a 5′ end of the target nucleic acid) or as a “reverse primer”(which initiates nucleic acid synthesis from a 5′ end of the extensionproduct produced from synthesis initiated from the forward primer).Likewise, the second primer can be designed as a forward primer or areverse primer.

“Readout” means a parameter, or parameters, which are measured and/ordetected that can be converted to a number or value. In some contexts,readout may refer to an actual numerical representation of suchcollected or recorded data. For example, a readout of fluorescentintensity signals from a microarray is the address and fluorescenceintensity of a signal being generated at each hybridization site of themicroarray; thus, such a readout may be registered or stored in variousways, for example, as an image of the microarray, as a table of numbers,or the like.

“Solid support”, “support”, and “solid phase support” are usedinterchangeably and refer to a material or group of materials having arigid or semi-rigid surface or surfaces. In many embodiments, at leastone surface of the solid support will be substantially flat, although insome embodiments it may be desirable to physically separate synthesisregions for different compounds with, for example, wells, raisedregions, pins, etched trenches, or the like. According to otherembodiments, the solid support(s) will take the form of beads, resins,gels, microspheres, or other geometric configurations. Microarraysusually comprise at least one planar solid phase support, such as aglass microscope slide.

“Specific” or “specificity” in reference to the binding of one moleculeto another molecule, such as a labeled target sequence for a probe,means the recognition, contact, and formation of a stable complexbetween the two molecules, together with substantially less recognition,contact, or complex formation of that molecule with other molecules. Inone aspect, “specific” in reference to the binding of a first moleculeto a second molecule means that to the extent the first moleculerecognizes and forms a complex with another molecules in a reaction orsample, it forms the largest number of the complexes with the secondmolecule. Preferably, this largest number is at least fifty percent.Generally, molecules involved in a specific binding event have areas ontheir surfaces or in cavities giving rise to specific recognitionbetween the molecules binding to each other. Examples of specificbinding include antibody-antigen interactions, enzyme-substrateinteractions, formation of duplexes or triplexes among polynucleotidesand/or oligonucleotides, receptor-ligand interactions, and the like. Asused herein, “contact” in reference to specificity or specific bindingmeans two molecules are close enough that weak noncovalent chemicalinteractions, such as Van der Waal forces, hydrogen bonding,base-stacking interactions, ionic and hydrophobic interactions, and thelike, dominate the interaction of the molecules.

As used herein, the term “T_(m),” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. Several equations for calculating theTm of nucleic acids are well known in the art. As indicated by standardreferences, a simple estimate of the Tm value may be calculated by theequation. Tm=81.5+0.41 (% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (see e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization (1985). Other references(e.g., Allawi, H. T. & SantaLucia, J., Jr., Biochemistry 36, 10581-94(1997)) include alternative methods of computation which take structuraland environmental, as well as sequence characteristics into account forthe calculation of Tm.

“Sample” means a quantity of material from a biological, environmental,medical, or patient source in which detection, measurement, or labelingof target nucleic acids is sought. On the one hand it is meant toinclude a specimen or culture (e.g., microbiological cultures). On theother hand, it is meant to include both biological and environmentalsamples. A sample may include a specimen of synthetic origin. Biologicalsamples may be animal, including human, fluid, solid (e.g., stool) ortissue, as well as liquid and solid food and feed products andingredients such as dairy items, vegetables, meat and meat by-products,and waste. Biological samples may include materials taken from a patientincluding, but not limited to cultures, blood, saliva, cerebral spinalfluid, pleural fluid, milk, lymph, sputum, semen, needle aspirates, andthe like. Biological samples may be obtained from all of the variousfamilies of domestic animals, as well as feral or wild animals,including, but not limited to, such animals as ungulates, bear, fish,rodents, etc. Environmental samples include environmental material suchas surface matter, soil, water and industrial samples, as well assamples obtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items. These examplesare not to be construed as limiting the sample types applicable to thepresent invention.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely”,“only” and the like in connection with the recitation of claim elements,or the use of a “negative” limitation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for taggingnucleic acid sequence fragments, e.g., a set of nucleic acid sequencefragments from a single genome, with one or more unique members of acollection of oligonucleotide tags, or sequence tokens, which, in turn,can be identified using a variety of readout platforms. As a generalrule, a given sequence token is used once and only once in any tagsequence. In addition, the present invention also provides methods forusing the sequence tokens to efficiently determine variations innucleotide sequences in the associated nucleic acid sequence fragments.

Before the present invention is described, it is to be understood thatthis invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupercedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anucleic acid” includes a plurality of such nucleic acids and referenceto “the compound” includes reference to one or more compounds andequivalents thereof known to those skilled in the art, and so forth.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W. H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Overview

In general, the present invention provides sequence tokens that can beused as markers for use in tagging nucleic acid sequences to allow forhigh throughput screening of the associated nucleic acid sequences andto enable identification of the source of any particular nucleic acidsof interest. A set of sequence tokens is generally designed so that eachsequence token within the set has a unique sequence that is differentthan the other sequence tokens in the set and does not cross-hybridizewith the other sequence tokens in the set. Therefore, based on theuniqueness of the sequence, each sequence token can be detectedindependently of all the other sequence tokens in the set by a uniqueprobe. Similar to any other oligonucleotide sequence, a sequence tokenor a concatenation of two or more sequence tokens can be ligated to DNAand can be used as a unique identification and sorting tag for theparticular associated DNA.

As described in greater detail below, the present invention is based onthe principle that a single unique sequence token is used only once totag a subgroup of a population of nucleic acid sequences. For example,if a population of nucleic acids includes 12 subgroups, 12 differentsequence tokens would be needed so that each subgroup of nucleic acidsis tagged with a different sequence token. In some embodiments, apopulation of nucleic acids will include a mixture of genomic DNA from apopulation of samples (e.g., cell lines) or subjects, such as humansubjects. In such embodiments, all genomic DNA from a first sample orsubject is tagged with a first unique sequence token and all the genomicDNA from a second sample or subject is tagged with a second uniquesequence token.

In such embodiments, all genomic DNA from a first sample or subject canbe digested into approximately 1 kilobase (kb) fragments and to allnucleic acid fragments from the first individual a first unique sequencetoken can be ligated as described below while all genomic DNA of asecond sample of subject can digested into approximately 1 kb fragmentsand to and to all nucleic acid fragments from the second individual asecond unique sequence token can be ligated. This process can berepeated for genomic DNA from a whole population of samples or subject.

As a result of the tagging process, the genomic DNA fragments from eachsample or subject in the population would be tagged with a uniquesequence token such that the particular sequence token used for any onesample or subject is not used for any other sample or subject in thepopulation. Therefore, once all the genomic DNA fragments from thesamples or subjects in the population are tagged with sequence tokens,the collection of tagged nucleic acids can be pooled and analyzed. Thepooled nucleic acids can subsequently be sorted based on the uniquesequence tokens. In addition, based on the uniqueness of the sequencetoken tags, any one tagged nucleic acid fragment can be identified asbelonging to any one original sample or subject based on the uniquesequence token tag used to tag all the genomic DNA fragments of theparticular sample or subject.

Like any other nucleic acid sequence, a sequence token can be added byligation to DNA and can be used as a unique tag, or as part of a uniquetag. However, unlike conventional combinatorial tags, e.g. Brenner etal, Proc. Natl. Acad. Sci., 97: 1665-1670 (2000), where the position ofan elementary unit is important, in the present invention the locationof addition of a sequence token does not matter. For example, in aselection or generation process in accordance with the presentinvention, the sequence tokens can be grouped or added in any order,anywhere, even in branched structures. In an exemplary process describedbelow, it is only a matter of convenience that the sequence tokens areaffixed to the sample in a sequential manner and that their order mayreflect the original sample identity of the tagged polynucleotide beinganalyzed within a larger tagged population of sample.

Sequence tokens can be used to label a sample at one particular positionwithin the sample or at multiple positions. Multiple positions areuseful in order to enable larger numbers of samples to be labeled moreefficiently. The number of particular labeling positions can be definedat the ‘base’ of such labeling. Each of the possible positions could belabeled by any one of a unique set of tokens and the tokens for any onesuch set are different from those tokens that comprise each of the othersets. The number of tokens in each set can be defined as the‘dimensionality’ of the process. Thus, if one had 32 unique sequencetokens divided into 4 sets corresponding to 4 possible labelingpositions, the base of such labeling would be 4 and the dimensionalitywould be 8 (which is. 32 divided by 4). The number of unique sequencetokens required to tag a population of individual DNAs depends on theinteger value of the base and the integer value of the dimensionality asshown below for tagging 4,096 individual genomes:

Base Dimensionality No. of Seq. Tokens 2 12 24 4 6 24 8 4 32 16 3 48 642 128 4,096 1 4,096

In general, the present system provides compositions and methods fortracking and identifying all DNA from a particular sample or subject ina mixed population. The system allows for parallel analysis of DNA froma whole population and the capability of specifically identifying whichindividuals carry a particular genetic variation of interest. As such,the system is particularly useful for identifying genetic variations atparticular locations in genomic DNA of a population of individuals andfor identifying the presence of any rare alleles as well as carriers ofthe rare alleles as described in greater detail below.

The following description provides guidance for making and using thecompositions of the invention, and for carrying out the methods of theinvention.

Constructing of a Set of Mutually Discriminatory Sequence Tokens

In one aspect, sequence tokens of the invention are designed so thatthere are no adjacent GC pairs. In general, the sequence tokens aregenerated from a set of units called “triplets.” Each triplet group hasfour members, wherein each member of the group is different from theother members by at least 2 out of 3 bases. Each triplet group has whatis referred to as a “dual” group, wherein every member of the dual groupdiffers from the original triplet group by at least 1 out of 3 bases.The properties of the dual triplets are the same as the original set.This has the effect that when a sequence composed out of one form isdefined, then its dual can immediately be included.

Triplets and methods for constructing sequence tokens from triplets aredescribed below. Sequences are designed to be as different as possiblefrom one another, i.e. mutually discriminatory, to ensure hybridizationoccurs or ensure that hybridization does not occur. The triplets aredefined as “s_(i)w_(j),” where s is G or C, w is A or T, and i and j areeither 0 or a positive integer and represent, respectively, the numberof s elements or w elements present in the triplet. The dual of anytriplet is characterized by appending an apostrophe, as exemplifiedbelow:

TABLE 1 Exemplary Triplet and Corresponding Dual Groups Triplet Dual 1.sww and sww′ CAA CAT GAT GAA CTT CTA GTA GTT 2. WSW and WSW′ TGA TGT AGTAGA TCT TCA ACA ACT 3. wws and wws′ TAG TAC AAC AAG TTC TTG ATG ATC 4.www and www′ TAA TAT AAT AAA TTT TTA ATA ATT 5. sws and sws CAG CAC GACGAG CTC CTG GTG GTCThe above exemplary triplets can be classified as either symmetric orasymmetric. For example, in symmetric triplets, such as wsw, www, andsws, the duals are the inverse complements of the correspondingtriplets, that is, sws and sws′ are complementary to each other in thesense that each member of sws has a complement in the set sws′. Inasymmetric triplets, sww and wws, sequences have been chosen such thatsww and wws′ are complementary as are sww′ and wws′.

As mentioned above, the differences between the members of the tripletsare designated in table 2 below as “<3>” for 3 out of 3 being different,“<2>” for at least 2 out of 3 being different, or “<1>” for at least 1out of 3 being different.

TABLE 2 Base Differences Between Triplet Groups sww wsw wws www sws sww<2> wsw <2> <2> wws <2> <2> <2> www <1> <1> <1> <2> sws <1> <3> <1> <2><2>From these triplets, a set of exemplary 9-base sequence tokens is chosenso that each member of the set has the same base composition w₆s₃ (i.e.,each sequence token includes 3 s elements and 6 w elements) with no ssconjunctions. Each 9-base sequence token is referred to herein as a“trio” as it is composed of three triplets. By “the same basecomposition” is meant that all the sequence tokens have the same numberof s elements and w elements, where s is G or C and w is A or T.

TABLE 3 Exemplary Set of Nine-base Sequence Tokens sws.wsw.wwwsww.sww.wsw sww.wsw.wsw wws.wsw.wws wws.wws.wsw sws.wws.www sww.sww.wwssww.wsw.wws sww.wws.wsw wws.wws.wws sws.www.sww wsw.sws.www wsw.wsw.swwsww.wws.wws wws.www.sws sws.www.wsw wsw.sww.sww wsw.wsw.wsw sww.www.swswww.sws.wsw sws.www.wws wsw.sww.wsw wsw.wsw.wws wsw.wws.wsw www.sws.wwssww.sws.www wsw.sww.wws wws.wsw.sww wsw.wws.wws www.sww.sws sww.sww.swwsww.wsw.sww wws.wsw.wsw wsw.www.sws www.wsw.swsAn exemplary set of thirty-five different trios are shown in Table 3.Concern about complementary sequences being included in the above set isunnecessary, because triplets have already been selected to avoid this.Thus, the components of sww triplet group (CAA, GAT, CTT, GTA) are notcomplementary to the components of wws triplet group (CAA, GAT, CTT,GTA). The components that are complementary to the sww triplet group arefound in the wws′ dual group.

There are four representations for each triplet and, therefore, for any9-base sequence token there are 4×4×4 (=64) potential sequences. Of the64 different potential sequences, each specific sequence differs fromthe other 63 sequences by at least two out of the nine bases. In theexemplary set of 9-base sequence tokens of Table 3, there are 35different trio sets, which means that there are 2,240 different 9-basesequences differing from each other by at least 2 out of 9 bases. Thecorresponding dual groups can be added and provisions can be made forsymmetry breaking, such as adding an “A” to each of the 9-base sequencetokens. This would give a total of 4,480 exemplary sequence tokens withhigh discrimination, 20%, which can be used as ligation codes.

A smaller subset of sequence tokens can also be selected such that eachmember differs from the other members by at least 4 out of the 9 bases.Such a selection can be made by examining the individual trios to selectthe subset which fits the requirement. The structure resembles that ofthe genetic code, and of the 64 sequences in each trio, a set is chosen,such that each trio has only one triplet in common with all the others.If the members of each triplet group are re-named as a, b, c, and d (forexample, the members of triplet group sww could be “a” for CAA, “b” forGAT, “c” for CTT, and “d” for GTA), equivalent to A, G, C, and T in thecode, then a subset obeying the rules is the following:

I. aaa abb acc add bab bbc bcd bda cac cbd cca cdb dad dba dcb ddcThere are four other exemplary ways to generate this subset, the otherthree being as follows:

II. baa bbb bcc bdd cab cbc ccd cda dac dbd dca ddb aad aba acb adc III.caa cbb ccc cdd dab dbc dcd dda aac abd aca adb bad bba bcb bdc IV. daadbb dcc ddd aab abc acd ada bac bbd bca bdb cad cba ccb cdcWithin each set, the trios differ from each other by 4 out of the 9bases; between sets the differences are 2 out of the 9 bases. As will bedescribed, there are advantages in using different subsets, particularlyto remove or minimize instances of complementary interactions betweensequence tokens.

To give an example, consider the trio sequence “wws.wsw.sww”. Theinverse complement of this sequence is a member of the same trio. Thetransform can be written:

→ wws.wsw.sww → wws.wsw.sww (wws.wsw.sww)′ (wws)′(wsw)′(sww)′           ←The inverse complement of a particular trio comprised of triplets istrio of the corresponding duals. Define the following representations.

wws sww′ sww wws′ wsw wsw′ a AAC GTT a′ a CTT AAG a′ a ACA TGT a′ b ATGCAT b′ b GAT ATC b′ b AGT ACT b′ c TAG CTA c′ c GTA TAC c′ c TCT AGA c′d TTC GAA d′ d CAA TTG d′ d TGA ACT d′The above are normal representations and their assigned inversecomplements, transforming x→x′ in the dual. A type I selection for thenormal representation is chosen and the particular trio “bcd”(wws.wsw.sww), which is defined below, is considered.

→  b   c   d ATG TCT CAAThe inverse complement, written below, is:

→  b   c   d ATG TCT CAA TAC AGA GTT b′  c′  d′         ←This follows the rule that the inverse complement of bcd, (bcd)′=d′c′b′=(dcb)′. It is noted that (dcb) is included in a type I arrangement,but is not present in any of the other three types. Therefore, a type IIrepresentation for the dual is chosen in which the perfect match is notfound. There are three sequences which match to some extent:

→ bcd ATG TCT CAA a′c′d′ TTG AGA GTT     ← b′d′d′ TAC TCA GTT     ←b′c′a′ TAC AGA GAA     ←These, the closest approximations, each contain two base mismatches andthe underlined triplets show wherein the mismatches occur in the inversecomplements. Shown below are the different approximations for bed in thefour types preserving the doublet c′d′←.

→ I bcd ATG TCT CAA I b′c′d′ TAC AGA GTT     ← II a′c′d′ TTG AGA GTT    ← III d′c′d′ AAG AGA GTT     ← IV c′c′d′ ATC AGA GTT     ←Two mismatches in nine bases are introduced in the duals chosen in thisway, which, as shown below, can be additionally enhanced.

The discussion above shows that 16 representations can be chosen foreach trio which differs from the other trios in the group by 4 out of 9bases. Furthermore, the same can be done for the dual representation andsets of 16 can be chosen in such a way that the duals do not containperfectly matched sequences complementary to the trios tokens. As seenlater, the two sets may therefore be used together. Now, a subset isselected from the set of 35 trios that will have the maximum distance of4 mismatches out of 9 bases.

Below are represented the trio sequences recording only the positions ofthe s residues. A set of 9 trios can be selected that differ from eachother in at least two out of the three positions of the s residues. Asexemplified below, token numbers 1, 3, and 7, each have an s at position3, but are different with respect to the two other position at which thes is present. For example, token number 1 has an s at positions 1, 3,and 6, while token number 7 has an s present at positions 3, 7, and 9.The neighbors differ in three positions, and appear on a diagonal. Thenumber of mismatches is twice the position differences (2×2=4); thusthese exemplary trios will differ in 4 out of the 9 bases, as can beseen from the sequences shown side by side below (A=sww, B=wsw, C=wws,D=www, and E=sws):

Token s residue Full sequence Triplet number positions representationrepresentation 1 1 3  6 sws.wws.www ECD 2  2 4  7 wsw.sww.sww BAA 3   35  8 wws.wsw.wsw CBB 4    4 6  9 www.sws.wws DEC 5 1   5 7 sww.wsw.swwABA 6  2   6 8 wsw.wws.wsw BCB 7   3   7 9 wws.www.sws CDE 8 1  4   8sww.sww.wsw AAB 9  2  5   9 wsw.wsw.wws BBC

Within each of the nine trios a set of 16 representations can beassigned, which differ from each other in 4 out of the 9 bases.Therefore, 9×16 (=144) 9-base sequence tokens can be selected with thisproperty. An equivalent set can be selected from the dual grouprepresentations. These differ from the normal representations in onebase out of three in each triplet and differ from the normal set in 3out of the 9 based. Since the duals contain sequences complementary tothe triplets, the complementarity can be minimized, as shown above, byselection of the appropriate subset. Moreover, an extra base can beadded to the right hand end, A in the case of the triplet (normal)representations, and T in the case of the dual representations. Thisadditional nucleotide breaks the symmetry even further but also adds abase difference. A total of 288 10-base sequence tokens can be definedin two sets of 144. Within each set the tokens differ by at least 4 outof 10 bases from each other (a discrimination of 40%). The two sets alsodiffer from each other in 4 out of 10 bases, and may be used together.

The system can be extended to larger sequences with the preservation oforthogonality. These are referred to as “quartets” and can be seen belowas an “s position” representation:

s residue Triplet positions representation 1 3  6       12a ECDCa  24  7      12a BAACa   3 5  8   11  a CBBBa    4 6  9  11  a DECBa 1   57  10    a ABAAa  2   6 8 10    a BCBAa   3   7 9      g CDEDg1  4   8       g AABDg  2  5   9Note that only 4 of the five triplets, A, B, C, and D, are used (A=sww,B=wsw, C=wws, D=www) and that a “g” is added to the last two and an “a”is added to the rest to provide the correct base composition, since D iswww. A set of 128 quartet sequences can be defined, differing in 6 outof 13 bases from each other. A further 128 may be chosen from the duals:these also differ in 6 out of 13 bases from each other and if a “t” isadded instead of an “a” and “c” instead of “g,” then a difference of 5out of 13 bases is defined between the two sets. Choosing the quartetrepresentation is an extension of the triplets. On of the choices is asfollows:

aaaa abbd acac addb babb bbca bcdd bdac cacc cbdb ccaa cdbd dadd dbacdcbb ddcaQuintuplets can be built up in the same way as above. Using the letterdesignation of the triplets, it is noted that the differences betweenadjacent trios are 6 out of 9 bases, and 4 out of 9 bases elsewhere.

ECD BAA CBB DEC ABA BCB CDE AAB BBC ECD — BAA 6 — CBB 4 6 — DEC 4 4 6 —ABA 4 4 4 6 — BCB 4 4 4 4 6 — CDE 4 4 4 4 4 6 — AAB 4 4 4 4 4 4 6 — BBC6 4 4 4 4 4 4 6 —To achieve quartets with 8 differences between each member out of 15,duos must be added that have the same property, a difference of 2 out of6 bases for adjacent duos, and 4 out of 6 bases elsewhere. This can bedone in two ways but only 5 duos can be shown to obey the rule as shownbelow:

AA AC BC BB CB AA — AC 2 — BC 4 2 — BB 4 4 2 — CB 4 4 4 2 — AB 2 2 4 2 2ED 2 2 4 4 2 BA 2 4 2 2 4 DE 2 2 2 4 4 CC 4 2 2 4 2 AA AB BB BC CC AA —AB 2 — BB 4 2 — BC 4 4 2 — CC 4 4 4 2 — BA 2 4 2 2 4 DE 2 4 4 2 2 ED 2 24 4 2 CB 4 2 2 4 2 AC 2 2 4 2 2The tables show the matches of the residual duos to the selected sets.

In each case, residual duos can be arranged into a set of four obeyingthe same rule:

AB ED BA DE AB — ED 2 — BA 4 4 — DE 4 4 2 — CC 4 2 4 2 BA DE ED CB BA —DE 2 — ED 4 4 — CB 4 4 2 — AC 4 2 2 4Thus, two subsets of sequence tokens can be created, one with fivequartets, the other with 4 using either subset. There are 80 in thefirst, and 64 in the second. Each member of each subset will differ fromother members by 8 out of 15 bases; between the subsets, the minimumdifference is 6 out of 15. The quintets can be arranged to give 16 setsdiffering between each member in 8 of 15 bases. By the appropriatechoice of dual representations, the differences between subsets can beincreased to 8 of 15. Thus, at least 144 sequence tokens are availablewith differences amounting to 8 out of 15.

Attaching Sequence Token Tags to Polynucleotides

A general procedure for attaching oligonucleotide tags of the inventionto polynucleotides is illustrated in FIGS. 1A-1B. Polynucleotides (100)are generated that have overhanging ends (102), for example, bydigesting a sample, such as genomic DNA, cDNA, or the like, with arestriction endonuclease. Preferably, a restriction endonuclease is usedthat leaves a four-base 5′ overhang that can be filled-in by onenucleotide to render the fragments incapable of self-ligation. Forexample, digestion with Bgl II followed by an extension with a DNApolymerase in the presence of dGTP produces such ends. Next, to suchfragments, initial adaptors (106) are ligated (104). Initial adaptors(106) (i) attach a first segment, or word, of an oligonucleotide tag toboth ends of each fragment (100). Initial adaptors (106) also contain arecognition site for a type IIs restriction endonuclease that preferablyleaves a 5′ four base overhang and that is positioned so that itscleavage site corresponds to the position of the newly added segment.(Such cleavage allows segments to be added one-by-one by use of a set ofadaptor mixtures containing pairs of segments, or words). In one aspect,initial adaptor (106) is separately ligated to fragments (100) from eachdifferent sample, e.g. each different individual genome within apopulation.

In order to carry out enzymatic operations at only one end of adaptoredfragments (105), one of the two ends of each fragment is protected bymethylation and operations are carried out with enzymes sensitive to5-methyldeoxycytidine in their recognition sites. Adaptored fragments(105) are melted (108) after which primer (110) is annealed as shown andextended by a DNA polymerase in the presence of 5-methyldeoxycytidinetriphosphate and the other dNTPs to give hemi-methylated polynucleotide(112). Preferably, primer (110) has a capture moiety attached, such asbiotin, or the like. Polynucleotides (112) are then digested with arestriction endonuclease that is blocked by a methylated recognitionsite, e.g. Dpn II (which cleaves at a recognition site internal to theBgl II site and leaves the same overhang). Accordingly, such restrictionendonucleases must have a deoxycytidine in its recognition sequence andleave an overhanging end to facilitate the subsequent ligation ofadaptors. Digestion leaves fragment (112) with overhang (116) at onlyone end and free biotinylated fragments (113). After removal (118) ofbiotinylated fragments (113) (for example by affinity capture with beadshaving avidin molecules immobilized thereon), adaptor (120) may beligated to fragment (112) in order to introduce sequence elements, suchas primer binding sites, for an analytical operation, such assequencing, SNP detection, or the like. Such adaptor is convenientlylabeled with a capture moiety, such as biotin, for capture onto a solidphase support so that repeated cycles of ligation, cleavage, and washingcan be implemented for attaching segments of the oligonucleotide tags.After ligation of adaptor (120), a portion of initial adaptor (124) iscleaved so that overhang (126) is created that includes all (orsubstantially all) of the segment added by adaptor (106). After washingto remove fragment (124), a plurality of cycles (132) are carried out inwhich adaptors (130) containing pairs of segments, or individualsequence tokens, such as a triplet described above, are successivelyligated (134) to fragment (131) and cleaved (135) to leave an additionalsegment, or sequence token to produce. Such cycles are continued untilthe oligonucleotide tag (140) having a concatenated sequence ofindividual sequence tokens are complete, after which the taggedpolynucleotides may be subjected to analysis directly, or single strandsthereof may be melted from the solid phase support for analysis.

In certain embodiments, it is desirable to generate concatenatedsequence tokens comprising of two or more sequence tokens. This isparticularly desirable in embodiments in which the concatenated sequencetokens will be used as primer sequences for PCR or sequencing. In suchembodiments, the concatenated sequence tokens will have two uniquesequence tokens, with the two unit concatenated sequence token beingreferred to as a ditoken. Each unique sequence token of the ditoken willinclude 9 nucleotides with the ditoken having a total of 18 nucleotides.For example, the tokens of the Group I can be concatenated with thetokens of Group II to provide a set of 32 ditokens that are capable ofacting as hybridization sequences for PCR primers.

I. aaa abb acc add II. baa bbb bcc bdd bab bbc bcd bda cab cbc ccd cdacac cbd cca cdb dac dbd dca ddb dad dba dcb ddc aad aba acb adcAs will be appreciated one of skill in the art, the present applicationalso encompasses concatenated sequence tokens having three or moreunique sequence tokens, four or more unique sequence tokens and thelike.

In addition, the sequence tokens can also be separated by a linkersequence. A suitable linker sequence will generally be a nucleic acidsequence that is not contained in the sequence tokens. For example, thesequence tokens of Groups I and II above both do not contain a GCnucleotide sequence. Therefore, a suitable linker to use with Group Iand II sequence tokens is the Aci I restriction enzyme recognition site(CCGC). The advantage of this sequential ligation of tokens is to allowsuccessive combination of pools of genomes in the labeling processdescribed below, this is not stated explicitly and may be helpful tonote.

For example, the ditokens can be synthesized of two adaptors: a lefthand adaptor and a right hand adaptor, wherein each adaptor includesrestriction enzyme recognition sites that can be used to liberate thesequence token. Examples of sequence tokens and the left hand and righthand adaptors are provided below:

Left adaptor sequence:

Right adaptor sequence:

The oligonucleotides can be synthesized and then cloned into plasmidsfor validation by sequencing and then liberated using the correspondingrestriction enzymes and ligated together and added to nucleic acidfragments.

Detection of Rare Alleles

As noted above, the sequence tokens of the present invention can be usedto detect the presence or absence of rare alleles in a population. By“rare alleles” or “rare polymorphisms” is meant a mutation, including aninsertion, deletion or substitution, as well as a single nucleotidepolymorphism occurring at a low frequency in a population, such as atabout 0.1% to about 5%, including about 0.2% to about 4.5%, about 0.3%to about 4%, about 0.4% to about 3.5%, about 0.5% to about 3%, and thelike. Due to the low frequency of occurrence, rare alleles or raresingle nucleotide polymorphisms have traditionally been difficult toidentify. Unfortunately, conventional sequencing approaches do not havesufficient sensitivity to detect reliably rare alleles or mutantsequences in a pool of sequences when their abundances are less than afew percent. In order to have 95% confidence of identifying an allelewith a 2% frequency, 75 individuals would need to be sequenced. Inaddition, detection of these alleles assumes the ability to detectheterozygote peaks in a sequencing trace with good accuracy. Incontrast, the sequence tokens of the present invention permit theidentification of rare alleles and/or mutations in pools, or mixedpopulations, of nucleic acid sequences, such as a pool of genomicsequences, which is advantageous in several fields, including geneticsresearch, medical genetics, and oncology.

Prior to screening and detection of the presence or absence of a rareallele, the genomic DNA of population of individuals are each taggedwith a unique concatenation of sequence tokens to enable subsequentidentification and sorting of the population of DNA. However, based onthe guidance provided herein, one of skill in the art will appreciatethat a variety of unique tags can be constructed from the describedsequence tokens. A schematic of an exemplary tagged genomic DNA fragmentis provided in FIG. 2, panel A, and an exemplary concatenation processof sequence unique tokens is provided in FIG. 2, panel B.

As shown in FIG. 2A, the exemplary tagged genomic DNA fragment ofapproximately 1 kb includes a tag having a concatenation of four uniquesequence tokens separated by a first functional sequence (A) with thetag and genomic DNA fragment being flanked by second (B) and third (C)functional sequences. In general, while the tags will be variable, e.g.,unique, between the genomic DNA fragments of each individual, thefunctional sequences will be constant for all the genomic DNA fragmentsof all the individuals in the population. As used herein the “functionalsequences” can include a variety of sequences that facilitate subsequentanalysis and sorting, such as primer sequences, T7 RNA polymerasepromoter sequence, and restriction enzyme recognition sites. Forexample, in some embodiments, the second (B) and third (C) functionalsequences will include sequences complementary to forward and reverseprimers to facilitate amplification of the entire tagged genomic DNAfragment or to make single stranded copies of the entire tagged genomicDNA fragment for analysis. In addition, in some embodiments, the firstfunctional sequence (A) may also include a sequence complementary to aprimer that is different than the primer of the second (B) or third (C)functional sequences. As a result, this additional primer sequence willallow amplification of only the genomic DNA fragment flanked by thefirst (A) and third (C) functional sequences without amplification ofthe sequence token tag. In addition, the first (A) functional sequencecan also include a restriction enzyme recognition site that canfacilitate liberation of the sequence token tag from the genomic DNAfragment.

The exemplary tagging system shown in FIG. 2B is based on a 4×concatenation dimensionality with an 8 sequence token base at each levelto facilitate tagging of 4,096 (8×8×8×8=4096) individual genomes. Inthis system, a first set of 8 unique sequence tokens are used in thefirst position “P”, a second set of 8 unique sequence tokens are used inthe second position “Q”, a third set of 8 unique sequence tokens areused in the third position “R”, and a third set of 8 unique sequencetokens are used in the third position “S”. Therefore, this exemplarysystem requires a total of 32 individual sequence tokens (8+8+8+8=32).

In order to achieve the tagging of the genomic DNA, first the 4,096genomic DNA molecules are tagged in the first “P” position with thefirst set of 8 sequence tokens numbered 25 through 32 in repeatingsequential order from 25 to 32. For example, the genomic DNA fragmentsfrom individual number 1 would be tagged in the “P” position withsequence token number 25 and would be classified as P₂₅, the genomic DNAfragments from individual number 2 would be tagged in the “P” positionwith sequence token number 26 and would be classified as P₂₆, thegenomic DNA fragments from individual number 3 would be tagged in the“P” position with sequence token number 27 and would be classified asP₂₇, and so on though sequence token number 32 after which the taggingwould be repeated (e.g., the genomic DNA fragments from individualnumber 9 would be tagged in the “P” position with sequence token number25 and would be classified as P₂₅) until all the genomic DNA fragmentsfrom the 4,096 individuals or samples are tagged. Each set of P₂₅through P₃₂ tagged samples are then pooled into a single P₂₅₋₃₂ group toprovide 512 P₂₅₋₃₂ groups. For example, as shown in FIG. 2B, samplenumbers 1 though 8 are pooled into a first 512 P₂₅₋₃₂ group, samplenumbers 9 though 16 are pooled into a second 512 P₂₅₋₃₂ group, and etc.

The 512 P₂₅₋₃₂ groups are then tagged in the second “Q” position withthe second set of 8 sequence tokens numbered 17 through 24 in repeatingsequential order from 17 to 24. For example, the genomic DNA fragmentsfrom the first P₂₅₋₃₂ group would be tagged in the “Q” position withsequence token number 17 and would be classified as Q₁₇P₂₅₋₃₂, thegenomic DNA fragments from the second P₂₅₋₃₂ group would be tagged inthe “Q” position with sequence token number 18 and would be classifiedas Q₁₈P₂₅₋₃₂, the genomic DNA fragments from the third P₂₅₋₃₂ groupwould be tagged in the “Q” position with sequence token number 19 andwould be classified as Q₁₉P₂₅₋₃₂, and so on though sequence token number24 after which the tagging would be repeated (e.g., the genomic DNAfragments from the ninth P₂₅₋₃₂ group would be tagged in the “Q”position with sequence token number 17 and would be classified asQ₁₇P₂₅₋₃₂) until all the genomic DNA fragments from the 512 P₂₅₋₃₂groups are tagged. Each set of Q₁₇ through Q₂₄ tagged samples are thenpooled into a single Q₁₇₋₂₄P₂₅₋₃₂ group to provide 64 Q₁₇₋₂₄P₂₅₋₃₂groups. For example, as shown in FIG. 2B, sample groups 1 though 8 arepooled into a first 64 Q₁₇₋₂₄P₂₅₋₃₂ group, groups 9 though 16 are pooledinto a second 64 Q₁₇₋₂₄P₂₅₋₃₂ group, and etc.

The 64 Q₁₇₋₂₄P₂₅₋₃₂ groups are then tagged in the third “R” positionwith the third set of 8 sequence tokens numbered 9 through 16 inrepeating sequential order from 9 to 16. For example, the genomic DNAfragments from the first Q₁₇₋₂₄P₂₅₋₃₂ group would be tagged in the “R”position with sequence token number 9 and would be classified asR₉Q₁₇₋₂₄P₂₅₋₃₂, the genomic DNA fragments from the second Q₁₇₋₂₄P₂₅-32group would be tagged in the “R” position with sequence token number 10and would be classified as R₁₀Q₁₇₋₂₄Q₁₈P₂₅₋₃₂, the genomic DNA fragmentsfrom the third Q₁₇₋₂₄P₂₅₋₃₂ group would be tagged in the “R” positionwith sequence token number 11 and would be classified asR₁₁Q₁₇₋₂₄P₂₅₋₃₂, and so on though sequence token number 16 after whichthe tagging would be repeated (e.g., the genomic DNA fragments from theninth Q₁₇₋₂₄P₂₅₋₃₂ group would be tagged in the “R” position withsequence token number 9 and would be classified as R₉Q₁₇₋₂₄P₂₅₋₃₂) untilall the genomic DNA fragments from the 64 Q₁₇₋₂₄P₂₅₋₃₂ groups aretagged. Each set of R₉ through R₁₆ tagged samples are then pooled into asingle R₉₋₁₆Q₁₇₋₂₄P₂₅₋₃₂ group to provide 8 R₉₋₁₆Q₁₇₋₂₄P₂₅₋₃₂ groups.For example, as shown in FIG. 2B, sample groups 1 though 8 are pooledinto a first R₉₋₁₆Q₁₇₋₂₄P₂₅₋₃₂ group, groups 9 though 16 are pooled intoa second R₉₋₁₆Q₁₇₋₂₄P₂₅₋₃₂ group, and etc.

The 8 R₉₋₁₆Q₁₇₋₂₄P₂₅₋₃₂ groups are then tagged in the fourth “S”position with the fourth set of 8 sequence tokens numbered 1 through 8in repeating sequential order from 1 to 8. For example, the genomic DNAfragments from the first R₉₋₁₆Q₁₇₋₂₄P₂₅₋₃₂ group would be tagged in the“S” position with sequence token number 1 and would be classified asS₁R₉₋₁₆Q₁₇₋₂₄P₂₅₋₃₂, the genomic DNA fragments from the secondR₉₋₁₆Q₁₇₋₂₄P₂₅₋₃₂ group would be tagged in the “S” position withsequence token number 2 and would be classified asS₂R₉₋₁₆Q₁₇₋₂₄Q₁₈P₂₅₋₃₂, the genomic DNA fragments from the thirdR₉₋₁₆Q₁₇₋₂₄P₂₅₋₃₂ group would be tagged in the “S” position withsequence token number 3 and would be classified as S₃R₉₋₁₆Q₁₇₋₂₄P₂₅₋₃₂,and so on though sequence token number 8 until all the genomic DNAfragments from the 8 R₉₋₁₆Q₁₇₋₂₄P₂₅₋₃₂ groups are tagged. The taggedsamples are then pooled into a single S₁₋₈R₉₋₁₆Q₁₇₋₂₄P₂₅₋₃₂ pooledpopulation of uniquely tagged genomic DNA fragments from 4,096 differentsamples.

As a result of the unique sequence token tagging system the correctsample identification of any tagged genomic DNA fragment removed fromthe pooled population can be readily determined based on the sequencetokens at each of the S, R, Q, and P positions. For example, a genomicfragment tagged with sequence tokens S₁R₉Q₁₇P₂₉ is identified as beingfrom original sample number 5 (FIG. 2B).

FIG. 3 provides a schematic diagram of a method for identifyingindividuals or samples carrying a rare allele by using the sequencetoken tagging system. Prior to analysis of the tagged nucleic acidpopulation, a single stranded copy of all the tagged nucleic acids isproduced. The single strand copies of the tagged nucleic acid populationbe produced by, for example, using a priming sequence provided in thethird functional sequence (C) (FIG. 2A).

In certain embodiments in which the number of samples in a givenpopulation of tagged nucleic acids is high and/or the particular rareallele occurs at a low frequency, then it may be desirable to enrich thesample of tagged nucleic acids for only the tagged nucleic acidfragments that include the sequence of interest that carries the rareallele. This enrichment can be done by, for example, using a probe thatis complementary to a sequence of interest. For example, if the rareallele of interest is known to be present in a particular gene, such asthe p53 gene, an oligonucleotide probe can be used that is complementaryto a sequence found in the gene of interest to facilitate separation ofthe sequence token tagged fragments carrying the gene of interest byhybridization. As a result, the separation provides an enrichedpopulation of tagged nucleic acid fragments encoding the gene ofinterest in order to remove other sequence token tagged nucleic acidsthat may interfere in the subsequent analysis.

For example, in some embodiments, an oligonucleotide probe that iscomplementary to a fragment of interest can be constructed to include afirst member of a binding pair to facilitate separation of the taggednucleic acid fragments of interest. Exemplary binding pairs include, butare not limited to, biotin and avidin, biotin and streptavidin, and thelike. Other binding elements that can also be used to separate fragmentsof interest include magnetic beads, such as DYNABEADS™. In suchembodiments, the oligonucleotide probes are immobilized to the firstbinding member of the binding pair, such as biotin, avidin,streptavidin, or a magnetic bead, and the probes are then incubated withthe sample of tagged nucleic acid population under condition that allowhybridization between the oligonucleotide probes and the fragments ifinterest. Following an adequate amount of time, the hybridized probes aswell as the tagged fragments of interest can be separated from theremaining population of tagged nucleic acids using the second member ofthe binding pair, such as avidin or streptavidin if biotin is used, or amagnet if a magnetic bead is used.

In some embodiments, it may be desirable to perform further enrichmentselections to ensure only tagged nucleic acid encoding the fragment ofinterest are present in the enriched sample for the analysis. In suchembodiments, a second oligonucleotide probe can be used that iscomplementary to a second sequence on the fragment of interest that isdifferent than the sequence complementary to the first oligonucleotideprobe. The addition enrichment steps can be repeated as necessary toprovide the desired enrichment level to avoid contamination with taggednucleic acids that do not contain the fragment of interest. In yetanother embodiment, such fragments of interest could be rescued from themixture of all fragments by using two PCR primers (one of which islabeled with a specific ligand such as, but not restricted to, biotin)which amplify only the fragment of interest. The amplicon resultingcould then be isolated by binding of the ligand-bearing PCR fragment toa solid phase coated with its binding pair complement (which isstreptavidin in the case of biotin).

As shown in FIG. 3, a second type of enrichment step can also be carriedout in addition to the above-described separation step that provides forseparation the sample population based on highly frequent alleles thatmay also be present in close proximity to a rare allele that mayinterfere with identification of samples that contain the rare allele.Such “frequent alleles” or “frequent polymorphisms” include a mutation,such as an insertion, deletion, or substitution, as well as a singlenucleotide polymorphism occurring at a high frequency in a population,such as at about 5% or more, usually about 10% or more.

FIG. 4 provides a schematic depiction of how the tagged nucleic acidpopulation can first be separated into frequent allele groups prior toanalysis of the presence or absence of a rare allele. In the depictedexample, the rare allele of interest is known to exist at position 333;however, a frequent allele is also known to exist at position 301. Thetwo variants of the single nucleotide polymorphism of the frequentallele are a C at position 301 or a G at position 301. In such anembodiment, it would be desirable to divide the population of taggednucleic acids into two groups—the first group having a C at position 301and the second group having a G at position 301. The two differentgroups can then be analyzed for the presence or absence of the rareallele.

In some embodiments, an oligonucleotide primer can be used that iscomplementary to a sequence at least one nucleotide upstream of thefrequent polymorphism. The oligonucleotide primer and the tagged nucleicacids are incubated with a polymerase and necessary ddNTPs to provideextension of the oligonucleotide primer. In order to facilitateseparation, the ddNTP corresponding to one form of the frequent SNP canbe conjugated to a binding member (BM) and upon extension the particularddNTP-BM is incorporated into the oligonucleotide primer. The BM canthen be used to separate the two groups of the SNPs. The bindingmembers, as described above can be, for example, biotin, avidin,streptavidin, magnetic beads, and the like.

As shown in the example in FIG. 4, to separate the tagged nucleic acidsbased on the SNP occurring at position 301, an oligonucleotide primer isused that is complementary to a sequence terminating at position 300.The tagged nucleic acids and oligonucleotide, ddCTP and ddGTP-BM areincubated with a polymerase under conditions to allow incorporation ofthe nucleotides in to oligonucleotide primer. Based on either thepresence of G or C at position 301, either C or G-BM will beincorporated into the oligonucleotide primer. Following an adequateamount of time to allow extension, the population of heteroduplexedtagged nucleic acids and extended oligonucleotide probes are separatedbased on the binding moiety incorporated in the oligonucleotide probes.As a result, the tagged population will be divided into two groups—theC₃₀₁ group and the G₃₀₁ group. The two groups can then be separatelyanalyzed for the presence or absence of the rare allele.

Separation of the tagged nucleic acids containing the rare the allelecan be carried out in a similar manner to separation of the initial poolof tagged nucleic acids based on frequent alleles as described above. Inthe depicted example of FIG. 4, the rare allele of interest is known toexist at position 333. The two variants are either an A at position 333or a T at position 333 with the rare variant being the A at position333. In such an embodiment, the population of tagged nucleic acids wouldbe sorted to remove the tagged nucleic acids having the A at position333 and then identifying which samples the particular tagged nucleicacids originated from using the unique sequence token tags.

In some embodiments, an oligonucleotide primer can be used that iscomplementary to a sequence at least one nucleotide upstream of the rarepolymorphism present at a known position in the fragment. Theoligonucleotide primer and the tagged nucleic acids are incubated with apolymerase and necessary ddNTPs to provide extension of theoligonucleotide primer. In order to facilitate separation, the ddNTPcorresponding to the rare variant of the SNP is conjugated to a bindingmember (BM) and upon extension the particular dNTP-BM is incorporatedinto the oligonucleotide primer. The BM can then be used to separatetagged nucleic acids having the rare variant of the SNP from the taggednucleic acids that do not. The binding members, as described above canbe, for example, biotin, avidin, streptavidin, magnetic beads, and thelike. To separate the tagged nucleic acids based on the SNP occurring atposition 333, an oligonucleotide primer is used that is complementary toa sequence up position 332. The tagged nucleic acids andoligonucleotide, ddATP and ddTTP-BM are incubated with a polymeraseunder conditions to allow incorporation of the nucleotides in tooligonucleotide primer. Based on either the presence of T or the rare Aat position 333, either A or T-BM will be incorporated into theoligonucleotide primer. Following an adequate amount of time to allowextension, the population of heteroduplexed tagged nucleic acids andextended oligonucleotide probes are separated based on the bindingmoiety incorporated in the oligonucleotide probes. As a result, thetagged nucleic acids having the rare A polymorphism at position 333 canbe separated from the remaining tagged nucleic acids based on thebinding moiety and analyzed to determine which from original samples thetagged nucleic acids originate.

An alternative method of detecting a rare polymorphism is to formheteroduplex molecules using wild type RNA probes. A schematic diagramof the exemplary method is provided in FIG. 5. As in the example above,the whole population of sequence token tagged nucleic acids mayoptionally be enriched to provide the sequence token tagged nucleic acidfragments that include the sequence of interest that carries the rareallele. As described above, the enrichment step can be performed by, forexample, using labeled oligonucleotide probes complementary to a nucleicacid sequence known to exist in the nucleic acid fragment of interest.As a result, the separation provides an enriched population of taggednucleic acid fragments encoding the gene of interest in order to removeother sequence token tagged nucleic acids that may interfere in thesubsequent analysis. Following the optional enrichment step, singlestranded DNA copies of the sequence token tagged fragments of interestare made. In general, the single stranded DNA copies will include thenucleic acids fragments as well as the sequence token tags.

The enriched population of wild type RNA probes can be generated by, forexample, denaturization of the DNA, reannealing, nicking and digestionof mismatched DNA to remove all nucleic acid fragments having the rarepolymorphism and to provide a population of nucleic acids substantiallylacking the rare polymorphism. For example, the sequence token taggednucleic acids fragments of interest are treated with one or morerestriction enzymes to liberate the nucleic acid fragments from thesequence tokens and the functional sequences (e.g., functional sequencesA, B, and C (FIG. 2, panel A)). As noted above functional sequences Band C can be designed to incorporate restriction enzyme recognitionsites that

Alternatively, liberation of the nucleic acid fragments from thesequence tokens and the functional sequences can also be carried out byrepeated rounds of DNA amplification using primer sequencescomplementary to nucleic acid sequences in the functional sequences Band C (FIG. 2, panel A). Following amplification, the single strandedmaterial can be digested by treating the sample with a nuclease, such asSI nuclease.

After liberation of the nucleic acid fragments from the sequence tokentags, the nucleic acid fragments are subjected to denaturization of theDNA, reannealing, nicking and digestion of mismatched DNA to remove allnucleic acid fragments having the rare polymorphism. For example, thenucleic acids are denatured and reannealed to allow the nucleic acidfragments having the rare polymorphism to hybridize to nucleic acidfragments lacking the rare polymorphism. In general, the method is basedon the principle that since the nucleic acid fragments having the rarepolymorphism will be in a low concentration in the sample, such as onthe order of 0.1% to about 5%, the nucleic acid fragments not carryingthe rare polymorphism will be in excess and will drive the reactiontowards dilution of the rare polymorphism encoding nucleic acidfragments. Once the nucleic acid fragments have been allowed toreanneal, the mixture is treated with a nuclease, such as SI nuclease,that will digest any single stranded nucleic acids as well as nick andhydrolyze any mismatched double stranded nucleic acids. Any doublestranded nucleic acids having a first strand that includes the rarepolymorphism and a second strand that lacks the rare polymorphisms willresult in a mismatch at the rare polymorphism. As a result, these hybriddouble stranded nucleic acids will be nicked at the rare polymorphismmismatch and the both strands of the hybrid molecule will be hydrolyzed.Therefore, due to the denaturization, reannealing, nicking and digestionof mismatched DNA, the rare polymorphism containing nucleic acids(hybrid molecules) will be diluted out to provide a substantially purecomposition of nucleic acid fragments lacking the rare polymorphism. Thedenaturization, reannealing, and digestion of mismatched DNA can also beoptionally repeated to further dilute the rare polymorphism containingnucleic acids. Once a substantially pure population of nucleic acidfragments lacking the rare polymorphism is produced, the nucleic acidfragments are treated with a RNA polymerase to produce single strandedRNA probes of the nucleic acids lacking the rare polymorphism.

The single stranded RNA probes of the nucleic acids lacking the rarepolymorphism are then combined with the DNA copy of the enriched nucleicacid fragments, the mixture allowed to denature and anneal to formheteroduplexes of RNA and DNA (FIG. 4). Any double stranded heteroduplexmolecules in the composition having a DNA strand that includes the rarepolymorphism and a RNA strand lacking the rare polymorphisms will resultin a mismatch at the rare polymorphism. The heteroduplex molecules arethen treated with RNAse Ito nick the RNA molecules at the mismatch,remove the mismatch nucleotide, and produce a 3′ phosphate on the nickedRNA strand. The 3′ phosphate can then be removed using alkalinephosphatase and using a polymerase, such as T7 sequenase, BM-NTP isincorporated at the site of the nick. The BM can then be used toseparate tagged nucleic acids having the rare variant of the SNP fromthe tagged nucleic acids that do not. The binding members (BM), asdescribed above can be, for example, biotin, avidin, streptavidin,magnetic beads, and the like.

The identify of the tagged nucleic acids can be determined bysequentially sequencing the sequence tokens at each position beginningwith the S group, then the R, Q, and P groups. For example, a firstsequencing primer will be used that is complementary to the firstfunctional sequence (A, FIG. 2A) to determine the S position sequencetokens. Once the S position sequence tokens are determined, theremaining sequence tokens are also sequenced using sequencing primersthat are complementary to the upstream sequence token. For example, ifthe sequencing reveals that two types of S position sequence token arefound, e.g., S₁ and S₅, in the sorted population, then the second stepsequencing will utilize sequencing primers that are complementary to thesequences of the S₁ and S₅ sequence tokens. Once all the samples aresorted out, the positioning of the specific sequence tokens at eachposition S₁₋₈R₉₋₁₆Q₁₇₋₂₄P₂₅₋₃₂ are decoded to determine the identify ofthe samples encoding the rare alleles. For example, if the analysisreveal that the only sequence token present is S₁R₉Q₁₇P₂₉ then it can bedetermined that only sample number 5 contains the rare allele.

Sieving Devices

In one aspect, sorting of the sequence tokens tagged nucleic acidfragments can be carried out by using a series of gates represented bythe complements of the sequence tokens. Tags containing a particularsequence token would be stopped (e.g. by hybridization) at the gatecorresponding to its complement, all others would be let through. Sincethis would rely on 100% yield at each gate, this would not be anefficient use of sequence tokens. Instead, a sorting technique is usedwherein a collection of tagged DNAs would be divided into two groups,one group containing a particular sequence token, the other grouplacking the specific such token. An exemplary sorting technique suitablefor use with the present invention is the sorting technique described inBrenner, PCT publication number WO 2005/080604, which is incorporatedherein by reference. In the described sorting technique, populations canbe sorted independent of yield. Since oligonucleotide tags of theinvention do not include repeats of sequence tokens, they can be sortedin a binary sorting process as described below, which will be referredto herein as a “sieve.” As such, sequence tokens of a subset can beidentified by a sieving process, where a series of steps are carried outin which a subset (which may be in the form of a concatenation ofsequence tokens) is sorted at each step into those subsets that have aparticular sequence token and those that do not.

In particular, the device includes a reusable solid phase support suchas linear element, e.g., a pin, that carries a chemically stableanti-sequence token that is complementary to a specific sequence token,or a portion thereof in instances of concatenated sequence tokens,present in the population mixture. The chemically stable anti-sequencetoken can be a nucleic acid analogue sequence, such as a peptide nucleicacid, a nucleic acid with a non-charged backbone or a locked nucleicacid (LNA). This allows for an improved rate and specificity of thehybridization event with the sequence token. The linear element, such asthe pin, can be fabricated out of any suitable material, such asplastic, that will provide for desirable results and will not interfereswith the hybridization event. The linear element can be fabricated toeither indirectly or directly carry the anti-sequence token. Forexample, the sequence token may be immobilized directly to the surfaceor the linear element. Alternatively, the sequence token may beimmobilized to the surface of a secondary element, such as a glass orplastic beads that then attached to the linear element. In someembodiments, the linear elements may be constructed of fiber opticfragments that are attached directly or indirectly to a detection means.Anti-token sequences can be synthesized individually and thenimmobilized to either the linear element or the secondary element thatis then attached to the linear element.

In certain embodiments, the linear elements are arranged in a comb-likemanner, wherein each linear element has immobilized thereon ananti-token oligonucleotide that is complementary to a specific sequencetoken. The combs of linear elements may be further arranged andimmobilized in a block to provide an addressable array of linearelements, such as pins, wherein each linear element extending away fromthe block has immobilized thereon, either directly or indirectly asdescribed above, an anti-token oligonucleotide that is complementary toa specific sequence token. The block of addressable linear elements mayfurther be connected to solution pumping and delivery means, whereineach linear element and the respective immobilized anti-sequence tokenis brought into fluid communication with a solution containing thesequence token tagged nucleic acid fragments.

In further embodiments, the addressable block of linear elements may bepositioned in a robotic arm that is controlled by a programmable meansthat provides for movement of the addressable block from a firstposition to a second position, such as a first plate having channels ora plurality of wells, such as a first microliter plate, to a secondplate having channels or a plurality of wells, such as a secondmicroliter plate. In other embodiments, the addressable block remainsstationary while the plates are positioned in a robotic arm thatprovides for movement of the plates from a first position to a secondposition.

As noted above, the sieving device can be used to sort out at individualsteps a population of sequence token tagged genomes into those subsetsthat have a particular sequence token and those that do not. To carryout the methods using the sieving device, the individual genomes mustfirst be tagged with sequence tokens. For example, 16 individualsequence tokens in a 3× concatenation system can be used to tag 4,096(16×16×16=4096) individual genomes. In this system, a first set of 16unique sequence tokens are used in the first position “P”, a second setof 16 unique sequence tokens are used in the second position “Q”, and athird set of 16 unique sequence tokens are used in the third position“R”. Therefore, this exemplary system requires a total of 48 individualsequence tokens (16+16+16=48). Alternatively, 8 individual sequencetokens in a 4× concatenation system can also be used to tag 4,096(8×8×8×8=4096) individual genomes. This alternative exemplary systemrequires a total of 32 individual sequence tokens (8+8+8+8=32).

In order to achieve the tagging of the genomic DNA, first the 4,096genomic DNA molecules are sorted into 16 “P” groups, i.e., groups P₁,P₂, P₃, etc. The nucleic acids in each “P” group are tagged with thefirst set of 16 sequence tokens numbered 33 through 48. As such, thenucleic acids of group P₁ are all tagged with sequence token number 33,the nucleic acids of group P₂ are all tagged with sequence token number34, the nucleic acids of group P₃ are all tagged with sequence tokennumber 35, the nucleic acids of group P₄ are all tagged with sequencetoken number 36, etc. The “P” groups are then recombined and sortedagain into 16 “Q” groups, i.e., groups Q₁, Q₂, Q₃, etc. The nucleicacids in each “Q” group are tagged with the second set of 16 sequencetokens numbered 17 through 32. As such, the nucleic acids of group Q₁are all tagged with sequence token number 17, the nucleic acids of groupQ₂ are all tagged with sequence token number 18, the nucleic acids ofgroup P₃ are all tagged with sequence token number 19, the nucleic acidsof group Q₄ are all tagged with sequence token number 20, etc. The “Q”groups are then recombined and sorted again into 16 “R” groups, i.e.,groups R₁, R₂, R₃, etc. The nucleic acids in each “R” group are thentagged with the third set of 16 sequence tokens numbered 1 through 16.As such, the nucleic acids of group R₁ are all tagged with sequencetoken number 1, the nucleic acids of group R₂ are all tagged withsequence token number 2, the nucleic acids of group R₃ are all taggedwith sequence token number 3, the nucleic acids of group R₄ are alltagged with sequence token number 4, etc. A similar tagging system canalso be achieved by directly tagging all 4,096 genomes with 4,096different sequence tokens. As will be appreciated by one of skill in theart, the order of the sequence tokens is not significant. For example,sequence tokens 1 though 16 can be used for the P groups, sequencetokens 17 though 32 can be used for the Q groups, and the sequencetokens 33 though 48 can be used for the R groups.

In such an exemplary method, the sieving device would be fabricated tobe capable of separating the tagged nucleic acids into the 4,096individual genomes. The sieving device is particularly useful forallowing subgrouping and subsequent detection and identification ofindividuals carrying a genetic variation, such as a rare allele and/orsingle nucleotide polymorphism, against a large background of otherpolymorphisms. In carrying out the exemplary methods, a population oftagged nucleic acids is first sorted based on the sequence of thegenomic DNA to provider an enriched population of tagged genomic DNA.For example, a probe complementary to a nucleic acid sequence interest,such as a gene, can be used to select all tagged nucleic acids in apopulation that encode the nucleic acid sequence of interest. As aresult, the tagged genomic fragment from all individuals in thepopulation encoding the nucleic acid sequence of interest would besorted into an enriched population for further analysis as described ingreater detail above. Once the nucleic acids have been analyzed, thesieving device can be used to determine the identity of the individualsin a subgroup of tagged nucleic acids.

In general, the size of the final linear elements and the number ofmolecules that immobilized on each linear element will vary depending onthe sensitivity of the detection system. In general, the number ofmolecules immobilized to each linear element will be from about 10⁴molecules to about 10¹⁵ molecules or more, including about 10⁵ moleculesto about 10¹² molecules, such as about 10⁸ molecules. In certainembodiments, for example, the number of molecules capable of binding toeach linear element will be approximately 10⁸ molecules, which can beimmobilized to an area of between 2,500 μm² and about 10,000 μm² (anarea of approximately 50-100 μm by 50-100 μm) at a density ofapproximately one molecule per 50 to 100 Å². As such, the molecules maybe immobilized to beads or pins of approximately 10 to 100 μm diameter.The linear elements, such as pins will generally have a diameter ofapproximately 0.25 mm (250 μm) spaced apart by a similar distance, suchas 0.25 mm (250 μm).

Prior to use of the sieving device to determine the identity of theindividuals in a subgroup of tagged genomic DNA, the genomic DNA portionmay optionally be removed to avoid interference of sequences in thegenomic DNA with hybridization events between the sequence tokens andcomplementary anti-sequence tokens immobilized on the linear elements ofthe sieving device.

In order to effect the separation and sorting of the sequence tokens,the linear elements, or pins, can be arranges in a comb-like manner(FIG. 6, panel A) and arranged on a block in an addressable manner (FIG.6, panel B). For example a first comb having 16 linear elements willhave anti-sequence tokens complementary to sequence tokens 1 to 16immobilized on the linear elements in an addressable manner. Forexample, a first comb will include anti-sequence tokens complementary tosequence tokens 1 to 16 of the 16 “R” groups, i.e., groups R₁, R₂, R₃,etc., a second comb will include anti-sequence tokens complementary tosequence tokens 17 to 32 of the 16 “Q” groups, i.e., groups Q₁, Q₂, Q₃,etc., and a third comb will include ant-sequence tokens complementary tosequence tokens 33 to 48 of the 16 “P” groups, i.e., groups P₁, P₂, P₃,etc. FIG. 6, panel B, shows the bottom view of an exemplary structure ofa block having addressable combs. As a result, the block will haveanti-sequence tokens complementary to the 48 sequence tokens arranged inan addressable manner.

During use, each comb is positioned in a channel though which a solutioncontaining the sequence token is pumped thereby allowing each of thelinear elements of the comb to come into contact with the solution underconditions suitable for allowing hybridization to occur between thesequence tokens and the corresponding complementary anti-sequencetokens. Cross-sections of a comb placed in a channel are depicted inFIG. 6, panels C and D. As each pin comes into close contact with asequence token complementary to the anti-sequence token immobilizedthereon, hybridization occurs and the solution of sequence tokens isdivided among the linear elements on the comb. The comb is thentransferred to a series of wells containing a buffer solution, whereineach linear member is positioned in a different well. The temperaturesof the wells are then increased to allow denaturization of thehybridized sequence tokens into the separate wells. As a result of thefirst sieving process, the solution of sequence tokens is separated intoa plurality of groups based on the first sequence token position. Forexample, if using the system described above, the sample will be dividedinto the 16 “P” groups, i.e., groups P₁, P₂, P₃, etc., using theanti-sequence tokens immobilized on the linear elements that arecomplementary to sequence tokens 33 to 48. Each of the “P” group membersare then subjected to a second round of sorting using a “Q” specificcomb having the anti-sequence tokens immobilized on the linear elementsthat are complementary to sequence tokens 17 to 32. As a result, theinitial population of sequence tokens will be sorted into 256 “Q-P”subpopulations (16×16). Each of the 256 “Q-P” group members are thensubjected to a third round of sorting using a “R” specific comb havingthe anti-sequence tokens immobilized on the linear elements that arecomplementary to sequence tokens 1 to 16. As a result, the population ofsequence tokens will be sorted into 4,096 “R-Q-P” subpopulations(256×16). In certain embodiments, the combs used for the third or finalsorting step are fabricated of optic fibers that are capable oftransmitting a detectable signal to a receiving unit. In suchembodiments, the sequence tokens can be modified to carry a detectablemoiety, such as a fluorescent protein. As a result, the presence orabsence of the sequence token hybridized to the complementaryanti-sequence token immobilized at each linear element can be readilyassayed.

Kits and Systems

Also provided by the subject invention are kits for practicing thesubject methods, as described above, such as combs having an array ofimmobilized anti-sequence tokens each specific for a unique sequencetoken of a nucleic acid tag. In some embodiments, the kits containprogramming means to allow a robotic system to perform the subjectmethods, e.g., programming for instructing a robotic pipettor to add,mix and remove reagents, as described above. The various components ofthe kit may be present in separate containers or certain compatiblecomponents may be precombined into a single container, as desired.

The subject kits may also include one or more other reagents forpreparing or processing an oligonucleotide tag of sequence tokensaccording to the subject methods. The reagents may include one or morematrices, solvents, sample preparation reagents, buffers, desaltingreagents, enzymatic reagents, denaturing reagents, where calibrationstandards such as positive and negative controls may be provided aswell. As such, the kits may include one or more containers such as vialsor bottles, with each container containing a separate component forcarrying out a sample processing or preparing step and/or for carryingout one or more steps of a combinatorial library synthesis protocolsuing nucleic acid tags.

In addition to above-mentioned components, the subject kits typicallyfurther include instructions for using the components of the kit topractice the subject methods, e.g., to identify the presence or absenceof a rare allele using the subject sieving device according to thesubject methods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or subpackaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

In addition to the subject database, programming and instructions, thekits may also include one or more control analyte mixtures, e.g., two ormore control samples for use in testing the kit.

The above teachings are intended to illustrate the invention and do notby their details limit the scope of the claims of the invention. Whilepreferred illustrative embodiments of the present invention aredescribed, it will be apparent to one skilled in the art that variouschanges and modifications may be made therein without departing from theinvention, and it is intended in the appended claims to cover all suchchanges and modifications that fall within the true spirit and scope ofthe invention.

1-21. (canceled)
 22. A method of screening for the presence or absenceof a rare nucleotide allele of a polymorphism in multiple DNA samples,the method comprising the steps of: combining the following underhybridization conditions: (i) a mixture comprising single stranded DNAfragments derived from multiple DNA samples, wherein the single strandedDNA fragments comprise: (a) a unique tag corresponding to the DNA samplefrom which it is derived; and (b) a sequence of interest having apolymorphic site, the polymorphic site having a wild type nucleotideallele and a rare nucleotide allele; and (ii) RNA probes comprising asequence complementary to the sequence of interest having the wild typenucleotide allele at the polymorphic site; thereby forming RNA/DNAheteroduplexes; removing any mismatched nucleotide at the polymorphicsite from the RNA probes in the heteroduplexes; contacting theheteroduplexes with a nucleotide triphosphate complementary to the rarenucleotide allele under nucleic acid polymerization conditions;isolating heteroduplexes that have incorporated the nucleotidetriphosphate complementary to the rare nucleotide allele; anddetermining the unique tags of the single stranded DNA fragments of theisolated heteroduplexes to identify genomic DNA samples having the rarenucleotide allele of the polymorphism in the sequence of interest,thereby screening the multiple DNA samples for the presence or absenceof the rare polymorphism.
 23. The method of claim 22, wherein the uniquetag is a unique sequence token tag.
 24. The method of claim 22, whereinthe nucleotide triphosphate complementary to the rare nucleotide allelecomprises a binding moiety.
 25. The method of claim 23, wherein theisolating step comprises contacting the heteroduplexes to a solidsupport having the binding partner of the binding moiety immobilizedthereon.
 26. The method of claim 25, wherein the binding moiety isbiotin and the binding partner is avidin or streptavidin.
 27. The methodof claim 22, wherein the determining step comprises contacting thesingle stranded DNA of the isolated heteroduplexes under hybridizationconditions to a solid phase support comprising immobilized anti-tagswherein each anti-tag is specific for one of the unique tags.
 28. Themethod of claim 27, wherein the solid phase support comprisingimmobilized anti-tags is a microarray.
 29. The method of claim 22,wherein the determining step comprises sequencing all or part of theunique tags of the single stranded DNA of the isolated heteroduplexes.30. The method of claim 22, wherein the removing step comprisescontacting the heteroduplexes to RNaseI followed by removing the 3′phosphate groups in RNA probes at the site of mismatch nucleotideremoval.
 31. The method of claim 30, wherein removing the 3′ phosphategroups comprises treating the heteroduplexes with alkaline phosphatase.32. The method of claim 22, wherein the mixture is produced by enrichingDNA fragments from the multiple DNA samples that contain the sequence ofinterest having the polymorphic site and generating single stranded DNAfragments from the enriched DNA fragments.
 33. The method of claim 22,wherein the multiple DNA samples are multiple genomic DNA samples. 34.The method of claim 22, wherein the RNA probes are generated by:combining DNA fragments enriched for the sequence of interest from themultiple DNA samples to form a combined sample, wherein the enriched DNAfragments are double stranded; subjecting the combined sample todenaturing and reannealing conditions to produce double stranded DNAhybrids; digesting the DNA hybrids having a mismatch; producing RNAprobes from the undigested DNA fragments.
 35. The method of claim 34,further comprising removing the unique tags from the enriched DNAfragments in the combined sample prior to the subjecting step.
 36. Themethod of claim 35, wherein removing the unique tags from the enrichedDNA fragments in the combined sample comprises contacting the enrichedDNA fragments with a restriction enzyme.
 37. The method of claim 35,wherein removing the unique tags from the enriched DNA fragments in thecombined sample comprises performing repeated rounds of DNAamplification using a primer pair to produce an amplified sample,wherein a first of the primers in the primer pair is complementary to aregion between the unique tags and the DNA fragment to which the uniquetags are attached.
 38. The method of claim 35, wherein the DNAamplification is a polymerase chain reaction (PCR).
 39. The method ofclaim 35, wherein single stranded DNA in the amplified sample is removedprior to the subjecting step.
 40. The method of claim 37, whereinremoving the single stranded DNA in the amplified sample comprisestreating the amplified sample with SI nuclease.
 41. The method of claim32, wherein the subjecting and digesting steps are repeated prior to theproducing step.